Structural disruption of BAF chromatin remodeller impairs neuroblastoma metastasis by reverting an invasiveness epigenomic program

Background Epigenetic programming during development is essential for determining cell lineages, and alterations in this programming contribute to the initiation of embryonal tumour development. In neuroblastoma, neural crest progenitors block their course of natural differentiation into sympathoadrenergic cells, leading to the development of aggressive and metastatic paediatric cancer. Research of the epigenetic regulators responsible for oncogenic epigenomic networks is crucial for developing new epigenetic-based therapies against these tumours. Mammalian switch/sucrose non-fermenting (mSWI/SNF) ATP-dependent chromatin remodelling complexes act genome-wide translating epigenetic signals into open chromatin states. The present study aimed to understand the contribution of mSWI/SNF to the oncogenic epigenomes of neuroblastoma and its potential as a therapeutic target. Methods Functional characterisation of the mSWI/SNF complexes was performed in neuroblastoma cells using proteomic approaches, loss-of-function experiments, transcriptome and chromatin accessibility analyses, and in vitro and in vivo assays. Results Neuroblastoma cells contain three main mSWI/SNF subtypes, but only BRG1-associated factor (BAF) complex disruption through silencing of its key structural subunits, ARID1A and ARID1B, impairs cell proliferation by promoting cell cycle blockade. Genome-wide chromatin remodelling and transcriptomic analyses revealed that BAF disruption results in the epigenetic repression of an extensive invasiveness-related expression program involving integrins, cadherins, and key mesenchymal regulators, thereby reducing adhesion to the extracellular matrix and the subsequent invasion in vitro and drastically inhibiting the initiation and growth of neuroblastoma metastasis in vivo. Conclusions We report a novel ATPase-independent role for the BAF complex in maintaining an epigenomic program that allows neuroblastoma invasiveness and metastasis, urging for the development of new BAF pharmacological structural disruptors for therapeutic exploitation in metastatic neuroblastoma. Supplementary Information The online version contains supplementary material available at 10.1186/s12943-022-01643-4.

For nuclear extract preparation, cells were resuspended in subcellular fractionation buffer (described in Co-immunoprecipitation section), incubated for 20 minutes on ice before centrifugation at 720 xG for 5 minutes, and nuclei lysed with RIPA buffer. In the case of frozen tissues, pieces of 2 to 5 mm diameter were immersed in 200 to 500 μL of the same RIPA buffer, and each fragment was disrupted with 20 ceramic beads with a Bead Ruptor 12 (Omni, Kennesaw, GA, USA), using one cycle of agitation of 20 seconds at a speed of 5 m/s. In both cases, samples were incubated for 20 minutes on ice to allow cell lysis and cleared by centrifugation at maximum speed (i.e. 13,300 rpm) at 4ºC for 15 minutes. Supernatant fraction was kept and protein concentration quantified with DC protein assay (Bio-Rad, Hercules, CA, USA). A total of 30 μg of each protein sample were heated at 70ºC for 10 minutes, resolved onto precasted NuPAGE 4-12% Bis-Tris polyacrylamide gels (Invitrogen) and posteriorly transferred onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare Life Sciences). Membranes were blocked for 1 hour in 5% Albumin Bovine Fraction V (BSA; NZYTech, Lisbon, Portugal) or 5% non-fat dried milk (PanReac AppliChem ITW Reagents, Castellar del Vallès, Spain) diluted in tris-buffered saline 0.1% Tween 20 (Sigma-Aldrich) (TBS-T) and incubated overnight incubation with primary antibodies diluted in 5% BSA or 5% milk TBS-T (Table S10). Membranes were then incubated for 1 hour with horseradish peroxidase (HRP)-conjugated secondary antibodies and developed with Enhanced Chemiluminescence (ECL) (GE Healthcare Life Sciences).

siRNA transfection
Sets of custom siRNA duplexes against each targeted gene (i.e. ARID1A and ARID1B) with [dT] [dT] overhangs were purchased from Sigma-Aldrich, based on validated target sequences extracted from literature (Table S9). Block-iT fluorescent siRNA control (Invitrogen) was used as negative control and to monitor transfection efficiency. Neuroblastoma cells (2.5 × 10 5 cells/mL) were transfected with 25 nM of the indicated siRNAs with Lipofectamine 2000, following the manufacturer's instructions.
When performing transfection experiments combining different siRNAs, 25 nM siRNA was used for each gene to maintain silencing performance, rising total siRNA concentration to 50 nM. Therefore, concentration of the negative control siRNA was doubled to 50 nM, to compensate doses. In the case of single inhibition in these experiments, negative control siRNA was added to the specific siRNA of each condition to equate siRNA concentrations to 50 nM in all the conditions. Proliferation assays with siRNA-transfected cells were performed by trypan blue exclusion assay using the Cell Counter EVE (NanoEntek, Seoul, South Korea).

RNA-Sequencing (extended)
RNA was extracted from 72 hours-transduced 2.5 × 10 5 SK-N-BE(2) in 6-well plates in biological triplicates by scraping in Qiazol lysis buffer (Qiagen, Hilden, Germany) and purification with miRNeasy mini extraction kit (Qiagen), following the manufacturer's instructions, with an additional in-column step of DNAse I treatment (Qiagen). Total RNA was eluted in 20 μL of nuclease-free water, fluorescently quantified using Qubit RNA HS Assay (Invitrogen) and quality checked by analysis with RNA 6000 Nano Assay on a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). All samples contained enough material (> 1 μg) of high RNA quality (RIN 10/10). Library preparation and sequencing was RNA-Seq reads were mapped against human reference genome (GRCh38) using STAR software version 2.5.3a [5] with ENCODE parameters. Genes were quantified using RSEM version 1.3.0 [6] with default parameters and annotation file from GENCODE version 34. Differential expression analysis was performed with DESeq2 v1.26.0 R package [7] using a Wald test to compare control and problem samples. Differentially expressed genes were those with p-value adjusted < 0.05 and absolute fold-change (FC) > 1.5, or more restrained thresholds, when indicated. Functional enrichment analysis of Hallmarks gene set collection from MSigDB database were performed using Gene Set Enrichment Analysis (GSEA) software [8,9]. Heatmaps were generated by normalizing the normalized counts of each gene by the average counts of the gene in all conditions, and log2 transformation. This value was represented in a colour gradient heatmap using Microsoft Office Excel (Microsoft Corporation, Redmond, WA, USA) and TM4's Multiple Experiment-Viewer MeV [10] softwares.

Cell cycle analysis
Cell cycle analysis was performed by the propodium iodide method. SK-N-BE(2) and SH-SY5Y transduced cells were fixed 96 h after transduction in 70% ice-cold ethanol overnight at -20°C, at a density of 10 6 cells/mL. Fixed cells were washed twice with PBS and resuspended in a staining solution containing 15 μg/mL propidium iodide (Sigma-Aldrich), 1.14 mM sodium citrate (Sigma-Aldrich), and 0.3 mg/mL RNase A (PanReac AppliChem ITW Reagents) in PBS at of 10 6 cells/mL. Cells were incubated at room temperature (20-25 °C) in the staining solution for at least 30 minutes prior to FACSCalibur flow cytometer analysis (BD Biosciences, Franklin Lakes, NJ, USA). Flow cytometry results were analysed using the FlowJo v10.8 Software (BD Biosciences).

Cell death assays
Apoptotic cell death was analysed by staining chromatin with Hoechst 33342 (Sigma-Aldrich).
Hoechst staining assays were performed on neuroblastoma living cells plated in 24-well plates (8 × 10 4 cells/well). Twenty-four hours after plating, cells were stained with 0.05 mg/ml Hoechst for 30 minutes at room temperature. Stained nuclei were observed and photographed under ultraviolet fluorescence microscopy.
CellTox Green Cytotoxicity Assay (Promega) kit was used for the determination of cell toxicity involving permeabilization of cell membrane and releasing of free genomic DNA. Transduced neuroblastoma cells were seeded on black opaque 96-well plates (2 × 10 4 cells/well). Twenty-four hours later, the CellTox reaction was performed on these same plates by addition of the fluorescent dye, following the manufacturer's protocol. Cell lysis solution included in the kit was used as a positive technical control of cell death. Fluorescence was measured using an Appliskan (Thermo Scientific) microplate reader. Fluorescence signal was normalized against each control.

ATAC-Sequencing (extended)
ATAC-Seq samples were processed simultaneously in triplicates as previously reported [11], with minor modifications. A total of 5 × 10 5 SK-N-BE(2) cells were obtained by trypsin digestion, washed with cold DPBS (Gibco) and lysed in fresh lysis buffer from [11]. For tagmentation, Tn5 E54K, L372P was expressed and purified from pETM11 vector as described in [12]. Tn5 (0.35 mg/mL) loading was done with linker oligonucleotides (Tn5ME-A/Tn5MErev and Tn5ME-B/Tn5MErev) as previously reported [12] for 60 minutes at 23ºC. Loaded Tn5 was purified with a 30 KDa Amicon Ultra 0.5 centrifugal unit (Sigma-Aldrich) column and diluted to a final concentration of 0.1 mg/ml with glycerol 25%. Tagmentation reaction was performed in a 50 μL reaction (25 uL tagmentation mix, 5 μL of nuclei (10 5 cells/uL), 5 μL of loaded Tn5 and, 15 μL of nuclease free water) for 60 minutes at 37 ºC followed by 5 mins at 80 ºC for heat inactivation. Immediately after samples were purified using ATAC-Seq analyses were performed with nf-core ATAC-seq pipeline v.1.2.1 [13] with default parameters except for the human reference genome (GRCh38.104), modified to include the sequences of the shRNAs used in each of the experimental conditions reference genome (Table S9).
Accessibility peaks of representative genomic regions were obtained with the Integrated Genome Viewer [14]. Differentially accessible peaks were determined by DESeq2 by comparing Non-Silencing Control to shRNA combinations (FDR < 0.01). Genes associated with differentially accessible peaks, by means of genomic proximity, were overlapped with differentially expressed genes obtained by RNA-Seq. Density plot and heatmap occupancy were obtained from the alignment files with plotHeatmap and plotProfile functions of the deepTools pipeline [15], using Galaxy platform [16], over the peaks of interest.

Immunofluorescence
Actin filaments were stained with the Phalloidin dye. A total of 2 × 10 5 cells per well were seeded in collagen-coated glass cover slips in 24-well plates and grown for 2 days. Next, cells were rinsed twice with PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Cells were washed three more times in PBS and incubated in glycine 0.1 M in PBS at room temperature for 5 minutes under soft agitation. After 2 more washes with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS at room temperature. Two more washes with PBS were performed prior blocking with 3% BSA in PBS for 60 minutes at room temperature under soft agitation. After one more wash with PBS, cover slips were incubated with the staining solution containing phalloidin-iFluor 594 (Abcam) diluted according manufacturer's instructions, monoclonal Anti-β-Tubulin−FITC (Sigma-Aldrich) 1000-times diluted and DAPI 10 µ/mL (Invitrogen) in 3% BSA in PBS, for 1 hour at room temperature under soft agitation, in a dark wet chamber. After 3 final washes with PBS, cover slips were mounted onto microscopy slides using ProLong Diamond Antifade Mountant (Invitrogen), and visualized with a ZEISS LSM 980 confocal microscope (Oberkochen, Germany). Ten random fields were acquired for each biological replicate and processed using ImageJ software. Number of cells per field was counted using DAPI staining of nuclei, and area stained with phalloidin and anti-tubulin was calculated. Tubulin-positive area percentage was used as a reference of the surface occupied by the main body of the cell, whereas area percentage of actin filaments protruding from this main body was considered as a quantification measure of stress-fiber protrusions. Thus, quantification of filamentous actin protrusions for each field was performed by calculating the percentage of tubulinfree phalloidin area per cell, by subtracting the tubulin area percentage to the phalloidin area percentage.

In vitro luciferase assay
Dual-Glo Luciferase Assay System (Promega) was used to measure the luciferase signal of neuroblastoma cells in vitro. Different numbers of FLUC-transduced SK-N-BE(2) cells (1.25, 2.5 and 5 × 10 5 per well) 120 hours after transduction were plated in white opaque 96-well plates. Cell lysis and luciferase reactions were performed following the manufacturer's recommendations.
Luminescence was measured using an Appliskan microplate reader. Luciferase signal was linearly correlated to the number of viable cells, confirming the reproducibility and feasibility of the technique and the results.

Immunohistochemistry
Mice livers were fixed in 10% formalin solution (Sigma-Aldrich) overnight, and washed twice with PBS before embedded in paraffin and sliced. Tissue sections were deparaffinized overnight at 60 °C and rehydrated using graded alcohols. Heat-induced antigen retrieval was performed using citrate buffer (pH 6, 4 min, 11 5°C) in a pressurized heating chamber. Chromogranin A primary antibody (Roche 760-2519, diluted 1:20) was incubated overnight at 4°C after blocking endogenous peroxidase.
Tissue sections were incubated with secondary antibody (Dako) for 30 min at room temperature, developed using diaminobenzidine (Dako), and counterstained using hematoxylin.

Neuroblastoma patient sample dataset analyses
K-means clustering module of the R2 genomics analyses and visualization platform (department of Oncogenomics, Academic Medical Center of the University of Amsterdam; http://r2.amc.nl) was used to generate two predictive gene-signatures, based on genes downregulated upon BAF inhibition. Two independent neuroblastoma patient cohorts were used for the study: SEQC (n = 498; GSE62564) [17] and Kocak (n = 649; GSE45547) [18]. To select gene-signatures, data obtained from RNA-Seq and ATAC-Seq analysis was used. For the first gene-signature, genes significantly downregulated in RNA-Seq data (log2FC < -1.5 and adjusted p-value < 0.001) in response to BAF depletion and included in the repressed cell cycle-related hallmarks (GSEA) were selected. For the second genesignature, genes repressed in RNA-Seq data (log2FC < -0.75 and adjusted p-value < 0.05), with associated chromatin repressive events assessed by ATAC-Seq, and included in the repressed cell cycle-related hallmarks were selected. Limited by the availability of probes in both datasets, for kmeans analysis the first gene-signature included 171 genes, and the second one included 26 genes.
In both cases the number of draws was set 10x10. Heatmaps represent z-score normalized expression. Kaplan Meier, based on the clusters, were generated using overall survival and the logrank test was performed to assess differences between groups. Univariate and multivariate Cox proportional hazard regression analyses were used to assess the prognostic significance of BAF score on overall survival. These statistical analyses were performed using the IBM SPSS 21 software.
In addition, based on the gene-signatures, a z-score was calculated for each patient (by using the R2 Heatmap module) to study the correlations with clinical data (INSS and Risk Group).